Abnormal surface expression of sialoglycans on B lymphocyte cell lines from patients with carbohydrate deficient glycoprotein syndrome I A (CDGS I A)

The carbohydrate-deficient glycoprotein syndromes (CDGS) are genetic, multisystemic diseases characterized by deficiencies in the glycosylation of many secretory glycoproteins, lysosomal enzymes, and possibly cell surface glycoproteins resulting in central nervous system abnormalities and frequent early death by infection. Here we examined whether membranous glycoconjugates of lymphocytes are affected by this disorder. For this, we analyzed cell surface-expressed sialoglycans of Epstein Barr virus (EBV)-transformed B cell lines derived from peripheral B lymphocytes of several patients with CDGS I A. These CDG-LCL (lymphoblastoid cell lines) expressed differentiation markers comparable to those of other EBV-transformed B cell lines. No apparent defects in the gross glycosylation process of defined complex glycosylated proteins such as the surface-expressed major histocompatibility complex class I glycoprotein or secreted immunoglobulin (IgM) were identified. However, using a novel flow cytometric enzyme assay to measure cell surface alpha2,6 sialylation on live cells we found that CDG-LCL express less alpha2,6 sialylated glycans in comparison to other EBV-transformed B cell lines. Also, CDG-LCL bound less of the B lymphocyte lectin CD22, specific for alpha2,6 sialylated lactosamines and known to modulate B cell receptor mediated signaling, as demonstrated by using a soluble CD22-immunoglobulin fusion protein in flow cytometry. CDG-LCL showed stronger surface staining with the monoclonal antibody 1B2 which detects a distinct group of surface-expressed lactosaminyl epitopes. After pretreatment with neuraminidase of Newcastle disease virus (NDVN) it became apparent that in CDG-LCL a significantly larger portion of the 1B2 epitopes was sialylated in alpha2,3 linkage as compared to other B cell lines. Intracellular alpha2,6 sialyltransferase activity as well as polymerase chain reaction products specific for four different sialyltransferases did not significantly differ in CDG-LCL as compared to other EBV-B cell lines. Differences in sialylation may be caused by the respective oligosaccharide core structures available for alpha2,6 or alpha2,3 sialylation in CDG-LCL. Therefore, lymphocytes derived from CDGS patients have distinct deviations in their surface-expressed lactosaminoglycan structures which may affect functions as exemplified by reduced interactions of CD22 with its ligands.     

Drug delivery system (DDS) for the use of antisense oligodeoxynucleotide phosphorothioate in controlling gene expression]

Subgroups of the B cell malignancies are known to be associated with Epstein-Barr virus (EBV) infection, especially in immunocompromised patients. These are fatal and refractory to conventional antineoplastic therapy. B cells are usually post-mitotic cells and even mitogen activated or transformed B cells have shown relative resistance against viral mediated gene transfer. To address this issue, we employed a replication-defective herpes simplex virus-1 (HSV-1) to mediate gene transfer into EBV-transformed B cells. The virus expresses the herpes simplex virus thymidine kinase (HSV-TK) and the E. coli lacZ reporter genes and is designated T0Z.1. We used the lymphoblastoid cell line SWEIG as a model for human EBV-related B cell malignancy. This cell line was established by in vitro EBV infection of primary human peripheral blood mononuclear cells. When SWEIG cells were infected with T0Z.1, X-gal staining revealed lacZ expression in more than 20% cells even at multiplicity of infection (MOI) as low as 1 and the expression persisted for at least one week. Ganciclovir (GCV) administration after T0Z.1 infection effectively decreased the number of the infected tumor cells in a dose-responsive manner. Viral toxicity was analyzed by cell proliferation assay (MTS assay) and found to be little even at 10 MOI infection. Three MOI of the virus yielded maximum antineoplastic effect and more than 50% tumor cells were killed by HSV-TK/GCV. These results suggest the potential utility of replication-defective HSV-1 for the treatment of EBV-related B cell malignancies.     

Pentacyclic oxindole alkaloids from Uncaria tomentosa induce human endothelial cells to release a lymphocyte-proliferation-regulating factor

In the present study we show that pentacyclic but not tetracyclic oxindole alkaloids from Uncoria tomentosa (Willd.) DC. (Rubiaceae) induced EA.hy926 endothelial cells to release some yet to be determined factor(s) into the supernatant; this factor was shown to significantly enhance proliferation of normal human resting or weakly activated B and T lymphocytes. In contrast, proliferation of normal human lymphoblasts and of both the human lymphoblastoid B cell line Raji and the human lymphoblastoid T cell line Jurkat was inhibited significantly while cell viability was not affected. Tetracyclic oxindole alkaloids dose-dependently reduce the activity of pentacyclic oxindole alkaloids on human endothelial cells.     

IS-RT-PCR assay detection of MT-MMP in a human breast cancer cell line

The effect of the host system on the pathogenicity, immunogenicity, and antigenicity of infectious bursal disease virus (IBDV) was investigated. One classic (SAL) and one variant strain (IN) of IBDV were passaged separately six times in three host systems, namely BGM-70 continuous cell line, primary chicken embryo fibroblast (CEF) cells, or embryonating chicken eggs (embryos) or one time in the bursa of Fabricius (BF) of specific-pathogen-free (SPF) chickens. Passage in BGM-70 cells or CEF cells resulted in loss of pathogenicity, but viruses passaged in embryos or BF maintained their pathogenicity. For the immunogenicity study, the viruses described above were used to prepare live and inactivated vaccines, containing 10(3) mean embryo infectious doses (EID50s) and 10(5) EID50s respectively. These vaccines induced different levels of protection. It was concluded that the antigen titration methodology employing embryonating chicken eggs was not suitable for titration of viruses propagated in other host systems because of varying degrees of adaptation and/or pathogenicity of the viruses resulting in variability in antigen mass of the tested vaccines. To test this assumption, an antigen-capture enzyme-linked immunosorbent assay was used as a titration system to compare the antigenicity of viruses propagated in BGM-70 cells or BF. Preparations containing similar antigen masses were inactivated then inoculated into two age groups of SPF chickens and antibody titers were monitored. During the experimental period, the geometric mean virus-neutralizing (VN) antibody titers of the vaccinated groups did not differ significantly (P > 0.05).     

Structure and function of a ganglioside receptor for porcine rotavirus

A ganglioside fraction isolated from pooled intestines from newborn to 4-week-old piglets, which we previously partially characterized and showed to specifically inhibit the binding of porcine rotavirus (OSU strain) to host cells (M. D. Rolsma, H. B. Gelberg, and M. S. Kuhlenschmidt, J. Virol. 68:258-268, 1994), was further purified and found to contain two major monosialogangliosides. Each ganglioside was purified to apparent homogeneity, and their carbohydrate structure was examined by high-pH anion-exchange chromatography coupled with pulsed amperometric detection and fast atom bombardment mass spectroscopy. Both gangliosides possessed a sialyllactose oligosaccharide moiety characteristic of GM3 gangliosides. Compositional analyses indicated that each ganglioside was composed of sialic acid, galactose, glucose, and sphingosine in approximately a 1:1:1:1 molar ratio. Each ganglioside differed, however, in the type of sialic acid residue it contained. An N-glycolylneuraminic acid (NeuGc) moiety was found in the more polar porcine GM3, whereas the less polar GM3 species contained N-acetylneuraminic acid (NeuAc). Both NeuGcGM3 and NeuAcGM3 displayed dose-dependent inhibition of virus binding to host cells. NeuGcGM3 was approximately two to three times more effective than NeuAcGM3 in blocking virus binding. Inhibition of binding occurred with as little as 400 pmol of NeuGcGM3/50 ng of virus (approximately 2 x 10(7) virions) and 2 x 10(6) cells/ml. Fifty percent inhibition of binding was achieved with 0.64 and 1.5 microM NeuGcGM3 and NeuAcGM3, respectively. The free oligosaccharides 3'- and 6'-sialyllactose inhibited binding 50% at millimolar concentrations, which were nearly 1,000 times the concentration of intact gangliosides required for the same degree of inhibition. Direct binding of infectious, triple-layer rotavirus particles, but not noninfectious, double-layered rotavirus particles, to NeuGcGM3 and NeuAcGM3 was demonstrated by using a thin-layer chromatographic overlay assay. NeuGcGM3 and NeuAcGM3 inhibited virus infectivity of MA-104 cells by 50% at concentrations of 3.97 and 9. 84 microM, respectively. NeuGcGM3 (700 nmol/g [dry weight] of intestine) was found to be the predominant enterocyte ganglioside (comprising 75% of the total lipid-bound sialic acid) in neonatal piglets, followed by NeuAcGM3 (200 nmol/g [dry weight] of intestine). NeuGcGM3 and NeuAcGM3 together comprised nearly 100% of the lipid-bound sialic acid in the neonatal intestine, but their quantities rapidly diminished during the first 5 weeks of life. These data support the hypothesis that porcine NeuGcGM3 and NeuAcGM3 are physiologically relevant receptors for porcine rotavirus (OSU strain). Further support for this hypothesis was obtained from virus binding studies using mutant or neuraminidase-treated cell lines. Lec-2 cells, a mutant clone of CHO cells characterized by a 90% reduction in sialyllation of its glycoconjugates, bound less than 5% of the virus compared to control cell binding. In contrast, Lec-1 cells, a mutant CHO clone characterized by a deficiency in glycosylation of N-linked oligosaccharides, still bound rotavirus. Furthermore, exogenous addition of NeuGcGM3 to the Lec-2 mutant cells restored their ability to bind rotavirus in amounts equivalent to that of their parent (CHO) cell line. In the virus-permissive MA-104 cell line, NeuGcGM3 was also able to partially restore rotavirus infectivity in neuraminidase-treated cells. These data suggest that gangliosides play a major role in recognition of host cells by porcine rotavirus (OSU strain).     

Growth arrest of Epstein-Barr virus immortalized B lymphocytes by adenovirus-delivered ribozymes

Epstein-Barr virus (EBV) infection is associated with several human diseases that involve unrestricted proliferation of B lymphocytes. EBV nuclear antigen 1 (EBNA-1) is expressed in all EBV-infected cells and plays an essential role in persistence of the EBV genome. EBNA-1 has also been reported to have oncogenic potential. As an approach for treating EBV infections, we examined the capacity of EBNA-1 ribozymes delivered by recombinant adenoviruses to suppress EBNA-1 expression and to block virus-induced B cell proliferation. In contrast to primary B cells, EBV-transformed B lymphoblastoid cell lines expressed alphav integrins, the adenovirus internalization receptors, and were also susceptible to adenovirus-mediated gene delivery. Adenovirus delivery of a specific ribozyme (RZ1) to lymphoblastoid cell lines, suppressed EBNA-1 mRNA and protein expression, significantly reduced the number of EBV genomes, and nearly abolished cell proliferation in low serum. Adenovirus delivery of RZ1 also prevented EBV infection of an established EBV-negative B cell line. These studies demonstrate the potential use of adenovirus-encoded ribozymes to treat EBV-induced lymphoproliferative disorders.     

Pentoxifylline prevention of altered hepatocyte calcium regulation during hemorrhagic shock/resuscitation

The role of immune complexes (Icx) in B-cell memory formation and affinity maturation allow for their potential use as vaccines. Recently, a new immune complex vaccine has been developed that is currently under field trials conducted in commercial poultry. This immune complex vaccine is developed by mixing live intermediate plus infectious bursal disease virus (IBDV) with hyperimmune IBDV chicken serum (IBDV-Icx vaccine). Here we have investigated the infectivity of this vaccine as well as the native IBDV (uncomplexed) vaccine in terms of differences in target organs, in target cells and speed of virus replication. At various days after inoculation on day 18 of incubation (in ovo) with either one dose of virus alone or the IBDV-Icx vaccine, the replication of IBDV and the frequency of B cells and other leucocyte populations were examined in the bursa of Fabricius, spleen, and thymus using immunocytochemistry. With both vaccines, IBDV was detected associated with B cells, macrophages and follicular dendritic cells (FDC) in bursa and spleen, although complexing IBDV with specific antibodies caused a delay in virus detection of about 5 days. Most remarkable was the low level of depletion of bursal and splenic B cells in IBDV-Icx vaccinated chickens. Furthermore, in ovo inoculation with the IBDV-Icx vaccine induced more germinal centres in the spleen and larger amounts of IBDV were localized on both splenic and bursal FDC. From these results we hypothesize that the working mechanism of the IBDV-Icx vaccine is related to its specific cellular interaction with FDC in spleen and bursa.     

Human lymphoblastoid cell line producing specific antibody against Rh-antigen D

A lymphoblastoid cell line producing specific anti-Rh antibody against D determinant was established by Epstein-Barr virus (EBV) infection. The lymphocytes were from an immunized male donor with high titre of anti-D antibody. The cells were preselected by rosetting them with Rh-positive erythrocytes before EBV infection. The cell line produced specific antibody of IgG class, namely IgG1 subclass, but it also produced nonspecific IgM and IgG. By rerosetting the cell line, the specific antibody-producing population could be enriched and the antibody titre increased in the culture supernatant to the level of immune sera.     

Effects of KW-3902, a selective and potent adenosine A1 receptor antagonist, on renal hemodynamics and urine formation in anesthetized dogs

Smyth line (SL) chickens develop a spontaneous, autoimmune, posthatch loss of pigment cells (vitiligo) in regenerating feather tissue. Smyth line vitiligo (SLV) is associated with lymphocyte infiltrations prior to and throughout the development of the disorder. It was the purpose of this study to determine the type, relative amounts, and proportions of pulp-infiltrating lymphocytes at various times throughout the growth of regenerating feathers. Feathers were plucked from 8-week-old chickens with and without SLV. Feather pulp cell suspensions were prepared when the regenerating feathers were 2, 3, 4, and 6 weeks of age. Cells were fluorescently labeled using a panel of mouse monoclonal antibodies specific for chicken lymphocytes. Both T and B cells infiltrated the feather pulp of chickens with SLV. T cell levels remained elevated throughout the 6 weeks of feather growth, while B cell levels steadily declined to control levels over the same time. The pulp-infiltrating cells were primarily T cells with an alphabeta T cell receptor expressing the Vbeta1 gene (TCR2+). The ratio between CD4+ and CD8+ cells was 1.42 and 0.75 in 2- and 6-week-old regenerating feathers from chickens with autoimmune SLV, respectively. In non-vitiliginous chickens this ratio was always near 1. These data suggest that TCR2+ T cells play an important role in SLV. CD4+ cells may play a recruiting/activating role, whereas CD8+ cells may have cytotoxic activity specifically directed against melanocytes. Additionally, this is the first report demonstrating the infiltration of B cells into the feather pulp of vitiliginous chickens. These B cells may directly/indirectly contribute to melanocyte destruction in SLV.     

Human Mig chemokine: biochemical and functional characterization

Mig is a chemokine of the CXC subfamily that was discovered by differential screening of a cDNA library prepared from lymphokine-activated macrophages. The mig gene is inducible in macrophages and in other cells in response to interferon (IFN)-gamma. We have transfected Chinese hamster ovary (CHO) cells with cDNA encoding human Mig and we have derived CHO cell lines from which we have purified recombinant human Mig (rHuMig). rHuMig induced the transient elevation of [Ca2+]i in human tumor-infiltrating T lymphocytes (TIL) and in cultured, activated human peripheral blood-derived lymphocytes. No responses were seen in human neutrophils, monocytes, or Epstein-Barr virus-transformed B lymphoblastoid cell lines. rHuMig was chemotactic for TIL by a modified Boyden chamber assay but rHuMig was not chemotactic for neutrophils or monocytes. The CHO cell lines, IFN-gamma-treated human peripheral-blood monocytes, and IFN-gamma-treated cells of the human monocytic cell line THP-1 all secreted multiple and identical HuMig species as revealed by SDS-PAGE. Using the CHO-derived rHuMig, we have shown that the species' heterogeneity is due to proteolytic cleavage at basic carboxy-terminal residues, and that the proteolysis occurs before and not after rHuMig secretion by the CHO cells. The major species of secreted rHuMig ranged from 78 to 103 amino acids in length, the latter corresponding to the full-length secreted protein predicted from the HuMig cDNA. Carboxy-terminal-truncated forms of rHuMig were of lower specific activity compared to full-length rHuMig in the calcium flux assay, and the truncated species did not block the activity of the full-length species. It is likely that HuMig plays a role in T cell trafficking and perhaps in other aspects of the physiology of activated T cells.     

Frequency and distribution of chromosomal integration sites of the Epstein-Barr virus genome

Human lymphocytes can be transformed in vitro by integration of the Epstein-Barr virus (EBV) into the host genome. To study whether integration sites are stable or changeable in the course of long-term cultivation, three EBV-transformed lymphoblastoid cell lines, along with positive control (Namalwa cell line) and negative control (noninfected lymphocytes) cells were studied. A biotinylated Bam H1WEBV-DNA fragment was used as the probe for fluorescence in situ hybridization to count the numbers and to localize the sites of integrated EBV-DNA. Among these cell lines, the percentage of cells with integrated signals varied from 58% to 91%, with a mean of 1.43 to 2.66 signals per cell. No consistent tendency of increasing or decreasing number of signals was observed for the three cell lines harvested at four different cultivation intervals (86-204 days after infection). Although the integration was not site-specific, high frequencies of integration did occur in all three cell lines at the following chromosomal sites: 1p31, 1q31, 2q32, 3q13, 6q24 and 7q31. Because there were multiple integration sites present, it was difficult to draw any conclusion on integration site stability.     

Infection of leukaemic B lymphocytes by Epstein Barr virus

Epstein-Barr virus (EBV) infection of B lymphocytes in vitro gives rise to immortalized lymphoblastoid cell lines. Previous reports have shown that chronic lymphocytic leukaemia (CLL) cells, although infectable by EBV, are resistant to immortalization (1-4), although a small number of CLL cell lines have been reported (5-7). In the present study we have analysed early events occurring after EBV infection in 16 CLL samples. Out of 16 samples, 15 could be infected by the virus and expressed the full EB viral nuclear antigen (EBNA) complex but only one out of 16 expressed the latent membrane protein (LMP). The five CLLs in which we could investigate the presence of viral episomes showed circularized EBV by 16 hours after infection. The sequence of EBNA expression and genome circularization mirrored that seen in normal B cells, although genome amplification was not detected. The only CLL sample which expressed LMP after EBV infection was induced to proliferate for 2-3 weeks, but no cell line was established. Immortalized cell lines were obtained from three out of 16 samples tested, but all were polyclonal for light chain expression and had arisen from the CD5-negative, normal B-cell population. Thus the inability of EBV to induce proliferation of most CLL cells correlated with the absence of LMP expression which is invariably expressed during immortalization of normal B cells. This novel type of restricted gene expression could be compatible with evasion of host immune responses and consequent long-term survival of the cell in vivo.     

Gene conversion in the chicken immunoglobulin locus: a paradigm of homologous recombination in higher eukaryotes

Gene conversion was first defined in yeast as a type of homologous recombination in which the donor sequence does not change. In chicken B cells, gene conversion builds the antigen receptor repertoire by introducing sequence diversity into the immunoglobulin genes. Immunoglobulin gene conversion continues at high frequency in an avian leukosis virus induced chicken B cell line. This cell line can be modified by homologous integration of transfected DNA constructs offering a model system for studying gene conversion in higher eukaryotes. In search for genes which might participate in chicken immunoglobulin gene conversion, we have identified chicken counterparts of the yeast RAD51, RAD52, and RAD54 genes. Disruption and overexpression of these genes in the chicken B cell line may clarify their role in gene conversion and gene targeting.     

Sensory epithelium of the vomeronasal organ express TrkA-like and epidermal growth factor receptor in adulthood. An immunohistochemical study in the horse

This study was undertaken to compare the genotoxic effects of arsenite in cultured human lymphocytes and lymphoblastoid cell lines from a group of normal human volunteers. The goal was to determine whether, as found with other genotoxins, subgroups might exist which showed relative high or low sensitivity to induction of sister chromatid exchanges (SCEs) by this metal. Primary lymphoblast cultures were established by treatment with phytohemagglutinin (PHA-L). Lymphoblastoid cell lines were established by transformation with Epstein-Barr virus. Cultures were exposed for 40 h to sodium arsenite (AsIII) and SCEs assayed by 5-bromo-2'-deoxyuridine incorporation and staining by fluorescence plus Giemsa. SCEs were increased by arsenite in a dose-dependent manner over the concentration range of 10(-7)-10(-5) M. SCEs could not be scored above 10(-5) M because of cytotoxicity. Comparison of SCE frequency in primary lymphocyte cultures among individuals showed substantial variation in sensitivity to arsenite, with some showing no significant effect while others showed a 2-3-fold increase in SCE frequency. In one lymphoblastoid cell line especially sensitive to arsenite, arsenic acid (AsV) or dimethylarsinic acid (DMA) at concentrations up to 10(-5) M did not increase the SCE frequency suggesting that AsIII is the active form of arsenic. When pooled data from the primary lymphocytes was compared to that obtained with the lymphoblastoid cells, the slopes of the dose-response curves for ASIII-induced SCEs were similar. The sensitivity of the majority of the individual primary lymphocyte cultures to SCE induction by arsenite was correlated with the sensitivity of the lymphoblastoid cultures established from the same individual. However, in three individuals no correlation was found. Individual lymphoblastoid cell lines retained their As sensitivity after cryopreservation and subsequent revival. Whether the genotoxic response to As is genetically controlled or the result of phenotypic selection is being explored in these stable lymphoblastoid cell lines.     

HLA-E surface expression depends on binding of TAP-dependent peptides derived from certain HLA class I signal sequences

Previous studies showed that HLA-E was expressed in lymphoblastoid cell line (LCL) 721.221 cells, but surface expression was lacking. To determine the signals controlling surface expression, we constructed a series of hybrid genes using complementary portions derived from the HLA-E and HLA-A2 genes. In this manner, a hybrid of HLA-E was identified, designated AEH, which differed from HLA-E by having the HLA-A2 signal sequence substituting for the HLA-E leader peptide. Transfection of LCL 721.221 cells with AEH induced HLA-E surface expression. Analysis of peptides bound to HLA-E revealed that a nonamer peptide derived from the A2 signal sequence was the predominant peptide bound. LCL 721.221 cells transfected with certain class I genes, including HLA-G, were also sufficient to promote peptide binding and HLA-E surface expression without increasing the level of HLA-E heavy chain synthesis. Peptides bound to HLA-E consisted of nine amino acids, with methionine at position 2 and leucine in the carboxyl-terminal position, and were nearly identical to the leader sequence-derived peptide previously shown to be a predominant peptide bound to the murine Qa-1 Ag. Signal peptides derived from certain HLA-B proteins with threonine in position 2 only marginally up-regulated HLA-E surface expression in .221 cells. An examination of HLA-E peptide binding in the TAP negative cell line .134 indicated that peptide binding to HLA-E was dependent on a functional TAP heterodimer regardless of whether peptide was available in cis, as in the AEH construct, or in trans, as in the class I transfectants of .221 cells.     

Peripheral type benzodiazepine receptor in T lymphocyte rich preparation

Some types of mood disorders and drugs are suggested to affect peripheral type benzodiazepine receptors (PBR), but their mechanisms are unclear. The isolation of pure lymphocytes is requisite for the investigation of the function of PBR on lymphocytes, since platelets and monocytes also have many PBR. The objective of this study was to establish a method of binding assay for PBR using pure T lymphocytes. Mononuclear cells and T lymphocytes were prepared by using a density gradient material and magnetic beads, respectively. The cells were analyzed using flow cytometry and a counting chamber. Binding studies were performed using T lymphocytes from 10 normal volunteers. The T lymphocytes were incubated with [3H]PK11195, harvested on glass fiber filters, and counted with a plate scintillation counter. The binding data were analyzed by the Scatchard method. With the magnetic bead technique, pure T cells were selected that contained only 1.5% monocytes and platelet/cell ratio of 1.4. The Scatchard plot of the data indicated that only one type of specific binding site was involved in the binding. The dissociation constant (Kd) was 3.8+/-1.3nM (mean+/-SD), and the Bmax was 379+/-124 fmol//10(6) cells (mean+/-SD). The density gradient- magnetic beads technique can be used as an appropriate method of preparation of T cells for PBR binding assay.     

Repression of the c-myb gene by WT1 protein in T and B cell lines

The c-myb gene is primarily expressed in immature hematopoietic cells, and it is overexpressed in many leukemias. We have investigated the role of negative regulatory sites in the c-myb promoter in the Molt-4 T cell line and in the DHL-9 B cell line. A potential binding site for either the EGR-1 or WT1 protein was identified by in vivo footprinting in the 5'-flanking region of c-myb in a region of negative regulatory activity in T cells. We showed by electrophoretic mobility shift assay and electrophoretic mobility shift assay Western that WT1, EGR-1, and Sp1 bound to this site. A mutation of this site which prevented protein binding increased the activity of the c-myb promoter by 2.5-fold. In the DHL-9 B cell line, this site was nonfunctional; however, we found a potential EGF-1/WT1 site located more 3' in a region of negative regulatory activity. We showed that WT1, EGR-1, and Sp1 bound to this site, and that mutation of this site increased the activity of the c-myb promoter by 3.2-fold. Cotransfection of a WT1 expression vector repressed the activity of the c-myb promoter in both cell lines, and this repression was relieved when the EGR-1/WT1 sites were removed. Cotransfection of either an EGR-1 or Sp1 expression vector had no significant effect on the activity of the c-myb promoter. We conclude that WT1 is a negative regulator of c-myb expression in both T and B cell lines.